Tracefinder v4

Relative quantitation of selected lipids in the human plasma pools was done using either TraceFinder v. 4.1 or SIEVE v. 2.1 (Thermo Fisher Scientific, Cambridge, MA). SIEVE analysis was as previously described (details in the supplemental section).
Peak areas of all potential LPEs and LPCs in plasma (based on the results of the interlaboratory report) was obtained using TraceFinder 4.1. In-brief, a compound library based on the precursor ion mass (mass error +/−5ppm) of all potential LPCs and LPEs was created within the software. TraceFinder data was then exported as a .csv file for further analysis.

After reconstitution in 60% acetonitrile spiked with internal standards, samples were analysed using HPLC and triple-quadruple mass spectrometry and tandem mass spectrometry. Specifically, the system consisted of a TSQ system (Thermo Fisher Scientific) in line with an electrospray source and a Vanquish (Thermo Fisher Scientific) UHPLC consisting of a binary pump, degasser and auto-sampler outfitted with a XBridge Amide column (Waters; dimensions, 3.0 mm × 50 mm and a particle size of 3.5 μm). The mobile phase A contained 95% (v/v) water, 5% (v/v) acetonitrile, 10 mM ammonium hydroxide, 10 mM ammonium acetate, pH 9.0; B was 100% acetonitrile. The gradient was as follows: 0 min, 15% A; 3 min, 45% A; 10 min, 60% A; 10.1–11 min, 75% A; 11.1 min, 15% A; 11.1–15 min, 15% A with a flow rate of 150 μl min⁻¹. In positive/negative polarity switching mode, the capillary of the electrospray ionization source was set to 300 °C, with sheath gas at 35 arbitrary units, auxiliary gas at 5 arbitrary units and the spray voltage at 3.5 kV. The selective reaction of the protonated precursor ion and the related product ions were monitored. The transitions are listed as follows: dopamine (+) 154→137, dopamine_D4(+) 158→141. The peak area was integrated. Data acquisition and analysis were carried out using Xcalibur v.4.1 and Tracefinder v.4.1, respectively (both from Thermo Fisher Scientific).

Data was analyzed in XCalibur v4.0 and/or Tracefinder v4.1 (Thermo). Statistical analysis and graph generation was conducted in GraphPad Prism v7.04 (GraphPad Software La Jolla, CA).

Samples were thawed and kept on ice throughout processing. Cell and fraction samples in 10% (w/v) trichloroacetic acid in water were sonicated for 12 × 0.5 s pulses, protein was pelleted by centrifugation at 17,000 ×g from 10 min at 4 °C. The supernatant was purified by solid-phase extraction using Oasis HLB 1cc (30 mg) SPE columns (Waters). Columns were washed with 1 mL methanol, equilibrated with 1 mL water, loaded with supernatant, desalted with 1 mL water, and eluted with 1 mL methanol containing 25 mM ammonium acetate. The purified extracts were evaporated to dryness under nitrogen then resuspended in 55 μl 5% (w/v) 5-sulfosalicylic acid in water.
Acyl-CoAs were measured by liquid chromatography-high resolution mass spectrometry. 5–10 μl of purified samples in 5% SSA were analyzed by injection of an Ultimate 3000 Quaternary UHPLC coupled to a Q Exactive Plus (Thermo Scientific) mass spectrometer in positive ESI mode using the settings described previously (Frey et al., 2016 (link)). Quantification of acyl-CoAs was via their MS2 fragments and the targeted masses used for isotopologue analysis are indicated in Table S3. Data were integrated using Tracefinder v4.1 (Thermo Scientific) software. Isotopic enrichment in tracing experiments was calculated by normalization to unlabeled control samples using the FluxFix calculator (Trefely et al., 2016 (link)).